Dirk Linke

Emergence of carbapenem-non-susceptible extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany.

The spread of Gram-negative bacteria with plasmid-borne extended-spectrum beta-lactamases (ESBLs) has become a worldwide problem. This study analysed a total of 366 ESBL-producing Enterobacteriaceae strains isolated from non-selected patient specimens at the university hospital of Tübingen in the period January 2003 to December 2007. Although the overall ESBL rate was comparatively low (1.6 %), the percentages of ESBL-producing Enterobacter spp. and Escherichia coli increased from 0.8 and 0.5 %, respectively, in 2003 to 4.6 and 3.8 % in 2007. In particular, the emergence was observed of one carbapenem-resistant ESBL-producing E. coli isolate and five carbapenem-non-susceptible ESBL-positive Klebsiella pneumoniae isolates, in two of which carbapenem resistance development was documented in vivo under a meropenem-containing antibiotic regime. The possible underlying mechanism for this carbapenem resistance in three of the K. pneumoniae isolates was loss of the Klebsiella porin channel protein OmpK36 as shown by PCR analysis. The remaining two K. pneumoniae isolates exhibited increased expression of a tripartite AcrAB-TolC efflux pump as demonstrated by SDS-PAGE and mass spectrometry analysis of bacterial outer-membrane extracts, which, in addition to other unknown mechanisms, may contribute towards increasing the carbapenem MIC values further. Carbapenem-non-susceptible ESBL isolates may pose a new problem in the future due to possible outbreak situations and limited antibiotic treatment options. Therefore, a systematic exploration of intestinal colonization with ESBL isolates should be reconsidered, at least for haemato-oncological departments from where four of the five carbapenem-non-susceptible ESBL isolates originated.

Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane

Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term ‘autotransport’ debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters.

Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals

Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly 13C- and 15N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.

Detergents: an overview.

Detergents are used in molecular biology laboratories every day. They are present in cell lysis buffers (e.g., in kits for plasmid isolation), in electrophoresis and blotting buffers, and, most importantly, they are used for cleaning laboratory glassware and the hands of the laboratory staff. For these routine applications, a detailed knowledge of detergent properties is not necessary-they just work. When it comes to the isolation and purification of membrane proteins, one cannot rely on routine protocols. Many membrane proteins are only stable in a small number of different detergent buffer systems, and worst of all, different membrane proteins prefer very different detergents. Unfortunately, detergent properties are considered the domain of colloid science or physical chemistry, and thus, while the available amount of physico-chemical data on detergents is astounding, this data is rarely compiled in a way that is useful to biochemists. The aim of this chapter is to provide an overview of the physical and chemical properties of detergents commonly used in membrane protein science and to explain how these properties can be exploited for protein purification.

Bartonella spp.: Throwing light on uncommon human infections

After 2 decades of Bartonella research, knowledge on transmission and pathology of these bacteria is still limited. Bartonella spp. have emerged to be important pathogens in human and veterinary medicine. For humans, B. henselae is considered to represent the most relevant zoonotic Bartonella species and is responsible for cat scratch disease, bacillary angiomatosis, and other disorders. Over the years, many Bartonella species have been isolated from humans, cats, dogs, and other mammals, and infections range from an asymptomatic state (e.g., animal-specific species) to even life-threatening diseases (e.g., Oroya fever). It is obvious that the analysis of pathogenicity mechanisms underlying Bartonella infections is needed to increase our understanding of how these pathogens adapt to their mammalian hosts resulting in acute or chronic diseases.

Duplication of fgfr1 Permits Fgf Signaling to Serve as a Target for Selection during Domestication

The genetic basis of morphological variation both within and between species has been a lasting question in evolutionary biology and one of considerable recent debate. It is thought that changes in postembryonic development leading to variations in adult form often serve as a basis for selection . Thus, we investigated the genetic basis of the development of adult structures in the zebrafish via a forward genetic approach and asked whether the genes and mechanisms found could be predictive of changes in other species. Here we describe the spiegeldanio (spd) zebrafish mutation, which leads to reduced scale formation in the adult. The affected gene is fibroblast growth factor receptor 1 (fgfr1), which is known to have an essential embryonic function in vertebrate development. We find that the zebrafish has two paralogs encoding Fgfr1 and show that they function redundantly during embryogenesis. However, only one paralog is required for formation of scales during juvenile development. Furthermore, we identify loss-of-function alleles changing the coding sequence of Fgfr1a1 that have been independently selected twice during the domestication of the carp (Cyprinus carpio). These findings provide evidence for the role for gene duplication in providing the raw material for generation of morphological diversity.

The Use of Detergents to Purify Membrane Proteins

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, or biomolecular NMR. Protocols in this unit include the isolation and solubilization of biological membranes, phase separation, and support protocols for detergent removal and detergent exchange using different methods. Curr. Protoc. Protein Sci. 53:4.8.1‐4.8.30.

Efficient subfractionation of gram-negative bacteria for proteomics studies.

Proteomics studies of pathogenic bacteria are an important basis for biomarker discovery and for the development of antimicrobial drugs and vaccines. Especially where vaccines are concerned, it is of great interest to explore which bacterial factors are exposed on the bacterial cell surface and thus can be directly accessed by the immune system. One crucial step in proteomics studies of bacteria is an efficient subfractionation of their cellular compartments. We set out to compare and improve different protocols for the fractionation of proteins from Gram-negative bacteria into outer membrane, cytoplasmic membrane, periplasmic, and cytosolic fractions, with a focus on the outer membrane. Overall, five methods were compared, three methods for the fast isolation of outer membrane proteins and two methods for the fractionation of each cellular compartment, using Escherichia coli BL21 as a model organism. Proteins from the different fractions were prepared for further mass spectrometric analysis by SDS gel electrophoresis and consecutive in-gel tryptic digestion. Most published subfractionation protocols were not explicitly developed for proteomics applications. Thus, we evaluated not only the separation quality of the five methods but also the suitability of the samples for mass spectrometric analysis. We could obtain high quality mass spectrometry data from one-dimensional SDS-PAGE, which greatly reduces experimental time and sample amount compared to two-dimensional electrophoresis methods. We then applied the most specific fractionation technique to different Gram-negative pathogens, showing that it is efficient in separating the subcellular proteomes independent of the species and that it is capable of producing high-quality proteomics data in electrospray ionization mass spectrometry.

C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA.

The Bam complex is a highly conserved multiprotein machine essential for the assembly of β‐barrel outer membrane proteins. It is composed of the essential outer membrane protein BamA and four outer membrane associated lipoproteins BamB–E. The Yersinia enterocolitica Adhesin A (YadA) is the prototype of trimeric auotransporter adhesins (TAAs), consisting of a head, stalk and a β‐barrel membrane anchor. To investigate the role of BamA in biogenesis of TAAs, we expressed YadA in a BamA‐depleted strain of Escherichia coli, which resulted in degradation of YadA. Yeast‐two‐hybrid experiments and immunofluorescence studies revealed that BamA and YadA interact directly and colocalize. As BamA recognizes the C‐terminus of OMPs, we exchanged the nine most C‐terminal amino acids of YadA. Substitution of the amino acids in position 1, 3 or 5 from the C‐terminus with glycine resulted in DegP‐dependent degradation of YadA. Despite degradation all YadA proteins assembled in the outer membrane. In summary we demonstrate that (i) BamA is essential for biogenesis of the TAA YadA, (ii) BamA interacts directly with YadA, (iii) the C‐terminal amino acid motif of YadA is important for the BamA‐dependent assembly and differs slightly compared with other OMPs, and (iv) BamA and YadA colocalize.

Phase separation in the isolation and purification of membrane proteins

Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.